Differences in the signaling pathways of α1A- and α1B-adrenoceptors are related to different endosomal targeting

Aims: To compare the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. Methods: Using CypHer5 technology and VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). Results and Conclusions: Constitutive as well as agonist-induced trafficking of α1A and α1B ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α1A-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α1B-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin

Rapid automatic segmentation of abnormal tissue in late gadolinium enhancement cardiovascular magnetic resonance images for improved management of long-standing persistent atrial fibrillation

Background: Atrial fibrillation (AF) is the most common heart rhythm disorder. In order for late Gd enhancement cardiovascular magnetic resonance (LGE CMR) to ameliorate the AF management, the ready availability of the accurate enhancement segmentation is required. However, the computer-aided segmentation of enhancement in LGE CMR of AF is still an open question. Additionally, the number of centres that have reported successful application of LGE CMR to guide clinical AF strategies remains low, while the debate on LGE CMR’s diagnostic ability for AF still holds. The aim of this study is to propose a method that reliably distinguishes enhanced (abnormal) from non-enhanced (healthy) tissue within the left atrial wall of (pre-ablation and 3 months post-ablation) LGE CMR data-sets from long-standing persistent AF patients studied at our centre. Methods: Enhancement segmentation was achieved by employing thresholds benchmarked against the statistics of the whole left atrial blood-pool (LABP). The test-set cross-validation mechanism was applied to determine the input feature representation and algorithm that best predict enhancement threshold levels. Results: Global normalized intensity threshold levels T PRE = 1 1/4 and T POST = 1 5/8 were found to segment enhancement in data-sets acquired pre-ablation and at 3 months post-ablation, respectively. The segmentation results were corroborated by using visual inspection of LGE CMR brightness levels and one endocardial bipolar voltage map. The measured extent of pre-ablation fibrosis fell within the normal range for the specific arrhythmia phenotype. 3D volume renderings of segmented post-ablation enhancement emulated the expected ablation lesion patterns. By comparing our technique with other related approaches that proposed different threshold levels (although they also relied on reference regions from within the LABP) for segmenting enhancement in LGE CMR data-sets of AF patients, we illustrated that the cut-off levels employed by other centres may not be usable for clinical studies performed in our centre. Conclusions: The proposed technique has great potential for successful employment in the AF management within our centre. It provides a highly desirable validation of the LGE CMR technique for AF studies. Inter-centre differences in the CMR acquisition protocol and image analysis strategy inevitably impede the selection of a universally optimal algorithm for segmentation of enhancement in AF studies